BIOACTIVITY AND CHEMICAL INVESTIGATIONS OF PERESKIA BLEO AND PERESKIA GRANDIFOLIA
SIM KAE SHIN
THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
FACULTY OF SCIENCE UNIVERSITY OF MALAYA
KUALA LUMPUR
2010
UNIVERSITI MALAYA
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ii BIOACTIVITY AND CHEMICAL INVESTIGATIONS OF
PERESKIA BLEO AND PERESKIA GRANDIFOLIA
ABSTRACT
Ethnopharmacological data has been one of the common useful criteria in drug discovery. Pereskia bleo and Pereskia grandifolia (Cactaceae), commonly known as
‘Jarum Tujuh Bilah’ in Malay and ‘Cak Sing Cam’ in Chinese have long been used as natural remedies in Malaysia. The experimental approach in the present study was based on bioassay-guided fractionation. The crude methanol and fractionated extracts of both Pereskia spp. were initially investigated for their biological activities such as antioxidant, antimicrobial and cytotoxic effect against five human cancer cell lines, namely nasopharyngeal epidermoid carcinoma cell line (KB), cervical carcinoma cell line (CasKi), colon carcinoma cell line (HCT 116), hormone-dependent breast carcinoma cell line (MCF7), lung carcinoma cell line (A549) and the non-cancer human fibroblast cell line (MRC-5) using in vitro cytotoxicity assay, in order to identify the bioactive extracts of both Pereskia spp.
The hexane and ethyl acetate extracts of both Pereskia spp. generally showed stronger antioxidant activities than the other extracts. Ethyl acetate extracts of both Pereskia spp. also showed some mild antimicrobial activities against the tested bacteria.
In the cytotoxicity assay, both Pereskia spp. exerted no damage to MRC-5 normal cells.
The ethyl acetate extracts of both Pereskia spp. in general gave higher inhibition and stimulation values against various cancerous cell lines compared to other extracts. The cell deaths of the selected cancer cells elicited by the cytotoxic active extracts of both Pereskia spp. were found to be apoptotic in nature based on a clear indication of DNA fragmentation, which is a hallmark of apoptosis. In addition, the LUX RT-qPCR [real-
iii time reverse transcriptase–polymerase chain reaction (RT-qPCR) using LUX (Light Upon eXtension) primers] analysis showed that apoptosis elicited by the cytotoxic extracts on selected cancer cells was mediated by p53, caspase-3 and c-myc activation in different expression levels.
Methyl palmitate, methyl linoleate, methyl α-linolenate and phytol were identified from the hexane extract of Pereskia bleo by GCMS analysis whilst methyl palmitate, methyl linoleate, methyl α-linolenate and methyl stearate were identified from the hexane extract of Pereskia grandifolia. From the results of the biological screenings, it is observed that the ethyl acetate extracts generally have stronger biological activities than other extracts. Further chemical investigations were thus directed to the ethyl acetate extracts of both Pereskia spp.
2,4-Di-tert-butylphenol (1), α-tocopherol (2), phytol (3), ß-sitosterol (4), dihydroactinidiolide (5) and a mixture of sterols containing campesterol (6), stigmasterol (7) and β-sitosterol (4) were isolated from Pereskia bleo whilst 2,4-di-tert- butylphenol (1), α-tocopherol (2), ß-sitosterol (4) and mixture containing methyl palmitate (9), methyl oleate (10), methyl stearate (11) and 2,4-di-tert-butylphenol (1) were isolated from Pereskia grandifolia. It is interesting to note that 2,4-ditert- butylphenol (1), α-tocopherol (2) and ß-sitosterol (4) were isolated from the ethyl acetate extracts of both Pereskia spp. The cytotoxic activities of the isolated constituents were evaluated against the above human cell lines and further studies on their mode of action suggested that these activities are connected with induction of apoptosis.
In addition, the toxicity of both Pereskia spp. was evaluated in vivo and were considered safe in acute oral toxicities in experimental mice. The screening of the
iv locally grown Pereskia bleo and Pereskia grandifolia indicated the presence of alkaloids, but the concentration was very low.
The findings of Pereskia bleo and Pereskia grandifolia in the present study provided scientific validation on the use of the leaves of both Pereskia spp. for the treatment of cancer. Further studies on the mutagenic and toxicity effect over a longer period of time involving detection of effects on vital organ functions should be carried out to ensure that the plants are safe for human consumption.
v KAJIAN BIOAKTIVITI DAN KIMIA
PERESKIA BLEO DAN PERESKIA GRANDIFOLIA
ABSTRAK
Data etnofarmakologi merupakan salah satu kriteria umum yang berguna dalam penemuan sesuatu ubat tertentu. Pereskia bleo dan Pereskia grandifolia (Cactaceae) yang biasa dikenali sebagai ‘Jarum Tujuh Bilah’ di kalangan orang Melayu dan ‘Cak Sing Cam’ di kalangan orang Cina telah lama digunakan sebagai jamu semulajadi di Malaysia. Eksperimen dalam penyelidikan ini dijalankan berdasarkan pemfraksian berpanduan bioasei. Kajian awal bioaktiviti ekstrak mentah serta fraksi daripada kedua- dua spesies Pereskia, seperti kesan antioksidan, antimikrob dan kesitotosikan telah dikaji untuk mengenalpasti ekstrak bioaktif bagi kedua-dua spesies tersebut.
Penyaringan aktiviti kesitotoksikan telah dijalankan menggunakan asei kesitotoksikan in vitro ke atas lima titisan sel karsinoma manusia, iaitu titisan sel karsinoma epidermoid nasofarinks (KB), titisan sel karsinoma serviks (CasKi), titisan sel karsinoma kolon (HCT 116), titisan sel karsinoma payu dara yang melibatkan hormon (MCF7), titisan sel karsinoma paru-paru (A549) serta titisan sel manusia bukan karsinoma (MRC-5).
Ekstrak heksana dan etil asetat bagi kedua-dua spesies Pereskia secara umumnya menunjukkan aktiviti antioksidan yang lebih kuat daripada ekstrak lain.
Ekstrak etil asetat bagi kedua-dua spesies Pereskia juga menunjukkan aktiviti antimikrob yang lemah pada bakteria yang dikaji. Bagi asei kesitotoksikan, ekstrak kedua-dua spesies Pereskia tidak aktif ke atas sel normal MRC-5. Ekstrak etil asetat kedua-dua spesies Pereskia secara umumnya menunjukkan nilai rintangan dan stimulasi yang lebih tinggi ke atas pelbagai titisan sel kanser berbanding dengan ekstrak
vi lain. Kematian kanser sel yang dirangsang oleh ekstrak aktif kedua-dua spesies Pereskia telah didapati mengaruh kematian sel secara apoptosis berdasarkan tanda fragmentasi DNA yang jelas. Fragmentasi DNA merupakan ciri utama apoptosis.
Tambahan lagi, analisis LUX RT-qPCR [real-time reverse transcriptase–polymerase chain reaction (RT-qPCR) using LUX (Light Upon eXtension) primers] menunjukkan apoptosis yang dirangsang oleh ekstrak aktif ke atas sel kanser tertentu adalah menerusi pengaktifan p53, caspase-3 dan c-myc pada tahap ekspresi yang berbeza.
Metil palmitat, metil linoleat, metil α-linolenat dan fitol telah dikenalpasti daripada ekstrak heksana Pereskia bleo melalui analisis GCMS manakala metil palmitat, metil linoleat, metil α-linolenat dan metil stearat telah dikenalpasti daripada ekstrak heksana Pereskia grandifolia. Daripada hasil penyaringan biologi, ekstrak etil asetat secara umumnya didapati mempunyai aktiviti biologi yang lebih kuat daripada ekstrak yang lain. Kajian kimia seterusnya ditumpukan kepada ekstrak etil asetat bagi kedua-dua spesies Pereskia.
2,4-Di-tert-butilfenol (1), α-tokoferol (2), fitol (3), ß-sitosterol (4), dihidroaktinidiolid (5) dan satu campuran sterol yang mengandungi kampesterol (6), stigmasterol (7) and β-sitosterol (4) telah dipisahkan dan dikenalpasti daripada Pereskia bleo sementara 2,4-di-tert-butilfenol (1), α-tokoferol (2), ß-sitosterol (4) dan satu campuran sebatian yang mengandungi metil palmitat (9), metil oleate (10), metil stearat (11) dan 2,4-di-tert-butilfenol (1) telah dipisahkan dan dikenalpasti daripada Pereskia grandifolia. Perlu diambil perhatian bahawa 2,4-di-tert-butilfenol (1), α-tokoferol (2) dan ß-sitosterol (4) wujud dalam ekstrak etil asetat kedua-dua spesies Pereskia. Kajian aktiviti kesitotosikan sebatian ke atas titisan sel kanser dan penyelidikan selanjutnya ke atas mod tindakan, mencadangkan aktiviti sitotosik sebatian adalah berkaitan dengan induksi apoptosis.
vii Di samping itu, ketoksikan kedua-dua spesies Pereskia telah dikaji secara in vivo dan hasil eksperimen ketoksikan akut yang dijalankan ke atas tikus menunjukkan ekstrak tumbuhan adalah selamat. Penyaringan untuk Pereskia bleo dan Pereskia grandifolia tempatan menunjukkan kehadiran sejumlah kecil alkaloid sahaja.
Hasil kajian terhadap Pereskia bleo dan Pereskia grandifolia ini memberi fakta saintifik yang sah dalam penggunaan daun kedua-dua Pereskia spesies untuk pengubatan kanser. Kajian mutagenik dan kesan ketoksikan pada organ penting untuk jangka yang lebih panjang perlu dijalankan untuk memastikan tumbuhan tersebut adalah selamat bagi penggunaan manusia.
.
viii ACKNOWLEDGEMENTS
I wish to express my sincere appreciation and gratitude to my supervisors, Datin Professor Dr. Sri Nurestri Abdul Malek from Institute of Biological Sciences, Faculty of Science and Datin Professor Dr. Norhanom Abdul Wahab from Centre for Foundation Studies in Science, for their continuous supervisions, advices, guidance and continuous encouragements throughout my project. I thank them especially for their patience and the time spent in discussing various approaches and results.
I would also like to express my acknowledgment and appreciation to Dato’
Professor Dr. Hashim Yaacob from International University College of Nursing for the selection of the plant species and his invaluable advices in publishing the findings.
My utmost appreciation to Associate Professor Dr. Kim Kah Hui from Department of Physiology, Faculty of Medicine for making available the facilities in his laboratory for the animal study. Special thanks to Mr V.T. Johgalingam for his willingness in teaching me basic toxicity research techniques.
My appreciation also goes to Associate Professor Koshy Philip from Institute of Biological Sciences, Faculty of Science for the use of laboratory facilities to carry out the antimicrobial screenings. I would also like to thank Mr Saravana Kumar for his guidance in antimicrobial screening work.
I would like to gratefully acknowledge Madam Hong Sok Lai and Mr Lee Guan Serm for their guidance and advice in chemical investigations. My acknowledgement is
ix also extended to Miss Syarifah Nur Syed Abdul Rahman and Miss Gowri Kanagasabapathy for their help, patience and motivation in carrying out the laboratory experiments.
My sincere appreciation to Miss Sujata Ramasamy for her assistance and generous encouragements in cell culture work. My gratitude towards Miss Wong Kah Hui, Miss Fathiah Abdullah, Miss Maizatul Azmah, Mr Mohd Azrul Mohd Sari, Miss Chan Pek Yue, Miss Lai Li Kuan, Miss Law Ing Kin and Miss Veronica Alicia Yap for their moral support and friendship in providing a good working environment.
My heartfelt gratitude to Mr Yong Yean Kong, Miss Tan Hong Yien, Miss Ong Lai Yee, Miss Teh Siew Phooi, Miss Lee Yeat Mei, Mr Dino Tan Bee Aik and Dr. Andrew Lim for all the help, guidance and patience in the gene expression work, and especially access to the facilities in the laboratory. Special thanks to Mr Yong Yean Kong for sharing all the knowledge in molecular genetics. He has been particularly helpful with suggestions on molecular studies for this work and has never been short of useful comments, ideas and solutions when approached with difficulties.
In addition, I am also very grateful to the staff of the Faculty of Science, Mr Ghana, Puan Latipah, Mr Asokan, Mr Teo, Pak Din, Encik Rufli and Encik Khamis for their support in carrying out the project.
Special thanks to Miss Chaw Sook Ting, Miss Pauline Ong, Miss Stephanie Lau Chiau Hwee and Miss Lim Siew Hua for invaluable moral support, encouragement and many useful comments on my work.
x Last but not least, my deepest appreciation and love to my husband, Dr Lai Kim Leng for his understanding and endless patience, as well as my parents, sisters and my family-in-law for their encouragements and support which has inspired me to accomplish this study.
I would like to acknowledge the Ministry of Higher Education (MOHE) and University of Malaya for the Skim Latihan Akademik IPTA (SLAI) award that kept me financially sound throughout the study period. This research project was supported by research funds from University of Malaya (PJP F0155/2005D, PPP PS056/2007C, PS241/2008C) and MOHE (FRGS FP040/2008C).
xi TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK v
ACKNOWLEDGEMENTS viii
TABLE OF CONTENTS xi
LIST OF TABLES xvii
LIST OF FIGURES xx
LIST OF APPENDICES xxv
LIST OF ABBREVIATIONS xxvii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 9
2.1 Natural products 9
2.2 Cancer 9
2.2.1 Carcinogens 10
2.2.2 Cell cycle 11
2.2.3 Carcinogenesis 12
2.3 Apoptosis in cancer 13
2.4 Natural products and defence against carcinogenesis 19 2.5 Natural products with conventional therapeutic modalities 20
2.6 The Cactaceae family 21
2.7 The Pereskia genus 22
2.7.1 The Pereskia bleo 24
2.7.2 The Pereskia grandifolia 28
2.8 Bioactivity assays 32
2.9 Antioxidant activity 33
2.9.1 Oxygen and singlet oxygen 33
2.9.2 Free radicals and Reactive Oxygen Species (ROS) 33 2.9.3 Cellular effects of oxidative stress and related diseases 34 2.9.4 Biological antioxidant defence mechanisms 35
2.9.5 Synthetic and natural antioxidants 36
2.9.6 Methods to determine antioxidant activities 38
2.10 Antimicrobial activity 39
xii
2.11 In vitro cytotoxic activity tests 42
2.11.1 In vitro testing system 42
2.11.2 In vitro cytotoxic activity testing 44 2.11.3 Methods to determine cytotoxic activity 45
2.12 Apoptosis screening and detection 45
2.12.1 Detection of morphological changes 46
2.12.2 Detection of DNA fragmentation 47
2.12.3 TUNEL assay 48
2.13 Determination of expression level of apoptotic-related genes 49
2.13.1 Introduction to gene expression 49
2.13.2 Importance of gene expression measurement 50
2.13.3 Apoptotic-related genes 51
2.13.4 Methods of mRNA quantification 54
2.13.5 RT-PCR quantification 55
(i) Conventional RT-PCR quantification 56
(ii) The RT-qPCR quantification 57
2.13.6 Fluorogenic LUX primer RT-qPCR assay 60
2.14 Acute oral toxicity assessment 64
3 MATERIALS AND METHODS 67
3.1 Plant materials 67
3.2 Extraction and fractionation of methanol extract of plant samples 67
3.3 Antioxidant activity 68
3.3.1 Folin-Ciocalteu method 68
3.3.2 Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals
70
3.3.3 Reducing power assay 73
3.3.4 ß-Carotene bleaching method 73
3.3.5 Statistical analysis 74
3.4 Antimicrobial activity 75
3.4.1 Preparation of agar and broth 75
3.4.2 Test microorganisms and microbial culture 75
3.4.3 Agar disc diffusion assay 76
3.4.4 Broth dilution assay 78
3.4.5 Statistical analysis 79
3.5 In vitro cytotoxicity assay 79
3.5.1 Cell lines and culture medium 79
3.5.2 In vitro neutral red cytotoxicity assay 80
3.5.3 Statistical analysis 82
3.6 Detection of DNA fragmentation (apoptosis) 82
xiii 3.7 Determination of the expression level of apoptotic-related genes 83
3.7.1 Preparation of quantitative standard (QSTD) for real-time quantification
84 3.7.2 Validation and optimization of qPCR assay using LUX
primers
91 3.7.3 Application of optimized qPCR assay to experiment set 99
3.7.4 Statistical analysis 102
3.8 Acute oral toxicity 104
3.8.1 Test species 104
3.8.2 Procedure of acute oral toxicity 105
3.9 Extraction, isolation and identification of chemical constituents from the bioactive extracts
106
3.9.1 Instrumentation 106
3.9.2 Column chromatography 107
3.9.3 Thin Layer Chromatography (TLC) 107
3.9.4 Preparative-Thin Layer Chromatography (Prep-TLC) 108 3.9.5 Identification of compounds in hexane extracts of P. bleo
and P. grandifolia using GCMS
108 3.9.6 Extraction and isolation of chemical constituents from the
bioactive ethyl acetate extract of P. bleo
109 3.9.7 Extraction and isolation of chemical constituents from the
bioactive ethyl acetate extract of P. grandifolia
114
3.10 Screening for alkaloids 118
3.10.1 Preparation of Wagner’s reagent 118
3.10.2 Preliminary alkaloidal test 118
3.10.3 Confirmatory test for alkaloids 119
3.10.4 Test for quaternary and/or amine oxide bases 119
4 RESULTS AND DISCUSSIONS 120
4.1 Extraction yield of P. bleo and P. grandifolia 120 4.2 Antioxidant activity of P. bleo and P. grandifolia extracts 122
4.2.1 Determination of reducing capacity of P. bleo and P.
grandifolia extracts using Folin-Ciocalteau method
122 4.2.2 Scavenging activity of P. bleo and P. grandifolia extracts
on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals
126 4.2.3 Reducing power assay of P. bleo and P. grandifolia
extracts
132 4.2.4 ß-Carotene bleaching activity of P. bleo and P.
grandifolia extracts
137 4.2.5 Comparison of antioxidant activity of P. bleo and P.
grandifolia extracts
141
4.3 Antimicrobial activity of P. bleo and P. grandifolia extracts 142 4.3.1 Agar disc diffusion assay of P. bleo and P. grandifolia
extracts
144
xiv
4.3.2 Broth dilution assay 147
(i) MIC and MBC of P. bleo extracts 147 (ii) MIC and MBC of P. grandifolia extracts 148 4.3.3 Comparison of antimicrobial activity of P. bleo and P.
grandifolia
150
4.4 In vitro neutral red cytotoxicity assay 151
4.4.1 Cytotoxic activity of P. bleo and P. grandifolia extracts 154 (i) Human nasopharyngeal epidermoid carcinoma cell
line (KB)
154 (ii) Human cervical carcinoma cell line (CasKi) 156 (iii) Human colon carcinoma cell line (HCT 116) 158 (iv) Hormone-dependent breast carcinoma cell line
(MCF7)
160
(v) Lung carcinoma cell line (A549) 162
(vi) Human fibroblast cell line (MRC-5) 164
4.4.2 Cytotoxic activity of doxorubicin 166
4.4.3 Comparison of cytotoxic activity of P. bleo and P.
grandifolia
168
4.5 Detection of DNA fragmentation 170
4.5.1 DeadEndTM Colorimetric Apoptosis Detection System (Promega)
171 4.5.2 Induction of apoptosis by the cytotoxic active extracts of
P. bleo and P. grandifolia on the selected human cancer cells
172
4.6 Determination of the expression level of apoptotic-related genes 173 4.6.1 The choice of using LUX RT-qPCR assay in gene
expression study
175 4.6.2 Validation and optimization of the LUX RT-qPCR assay 176
(i) Specificity test of LUX primers 177
(ii) Optimization of the concentrations of LUX primers and MgCl2
182 (iii) Optimization of the annealing temperature (TA) 182 (iv) Determination of the lowest detection limit and
sensitivity of the LUX RT-qPCR assay
183 4.6.3 Considerations for RT-qPCR procedures 185
(i) Reagents 185
(ii) Negative controls 185
(iii) Internal reference gene 186
4.6.4 Data analysis 187
4.6.5 Expression level of apoptosis-related genes in the cytotoxic active extract-treated cells
189 (i) Expression level of apoptosis-related genes in the
P. bleo cytotoxic active extract-treated cells
189 (ii) Expression level of apoptosis-related genes in the
P. grandifolia cytotoxic active extract-treated cells
193 (iii) Summary of expression level of apoptosis-related
genes in the Pereskia spp. cytotoxic active extract-
196
xv treated cells
4.7 Acute oral toxicity assessment of P. bleo and P. grandifolia crude extracts
197
4.8 Chemical constituents from the bioactive extracts of P. bleo and P.
grandifolia
199 4.8.1 Identification of compounds in hexane extract of P. bleo
using GCMS
200 4.8.2 Identification of compounds in hexane extract of P.
grandifolia using GCMS
204 4.8.3 Chemical constituents from the bioactive ethyl acetate
extract of P. bleo
207 (i) Structural determination of 2,4-ditert-butylphenol
(1)
209 (ii) Structural determination of α-tocopherol (2) 212 (iii) Structural determination of phytol (3) 214 (iv) Structural determination of ß-sitosterol (4) 215 (v) Structural determination of dihydroactinidiolide (5) 217 (vi) Structural determination of mixture of sterols
(mixture A)
218 4.8.4 Chemical constituents from the bioactive ethyl acetate
extract of P. grandifolia
221 (i) Structural determination of 2,4-ditert-butylphenol
(1)
223 (ii) Structural determination of α-tocopherol (2) 224 (iii) Structural determination of ß-sitosterol (4) 225 (iv) Structural determination of phytone (8) 226 (v) Structural determination of mixture B 227 4.9 Cytotoxic and apoptosis effects of chemical constituents isolated
from the bioactive ethyl acetate extracts of P. bleo and P.
grandifolia
229 4.9.1 Cytotoxic and apoptosis effects of 2,4-di-tert-butylphenol
(1)
230 (i) Cytotoxic activity of 2,4-di-tert-butylphenol (1) 230 (ii) Induction of apoptosis by 2,4-di-tert-butylphenol
(1) on selected cells
232 (iii) Expression level of apoptosis-related genes in 2,4-
di-tert-butylphenol (1)-treated cells
232 4.9.2 Cytotoxic and apoptosis effects of α-tocopherol (2) 237 (i) Cytotoxicity activity of α-tocopherol (2) 237 (ii) Induction of apoptosis by α-tocopherol (2) on
selected cells
238 (iii) Expression level of apoptosis-related genes in α-
tocopherol (2)-treated cells
239
4.9.3 Cytotoxic effect of phytol (3) 241
4.9.4 Cytotoxic effect of ß-sitosterol (4) 243 4.9.5 Cytotoxic and apoptosis effects of dihydroactinidiolide (5) 245 (i) Cytotoxic activity of dihydroactinidiolide (5) 245
xvi (ii) Induction of apoptosis by dihydroactinidiolide (5)
on HCT 116 cells
246 (iii) Expression level of apoptosis-related genes in
dihydroactinidiolide (5)-treated HCT 116 cells
246 4.9.6 Cytotoxic effect of mixture of sterols (mixture A) 247 4.9.7 Cytotoxic and apoptosis effects of phytone (8) 249 (i) Cytotoxic activity of phytone (8) 249 (ii) Induction of apoptosis by phytone (8) on selected
cells
250 (iii) Expression level of apoptosis-related genes in
phytone (8)-treated cells
250 4.9.8 Cytotoxic and apoptosis effects of mixture B 253 (i) Cytotoxic activity of mixture B 253 (ii) Induction of apoptosis by mixture B on selected
cells
255 (iii) Expression level of apoptosis-related genes in
mixture B-treated cells
255 4.9.9 Comparison of cytotoxic and apoptosis effects of
chemical constituents isolated from the bioactive extracts of P. bleo and P. grandifolia
257
4.10 Screening for alkaloids of P. bleo and P. grandifolia 261
5 CONCLUSION 263
REFERENCES 270
APPENDICES 297
xvii LIST OF TABLES
Table Page
2.1 Endpoints used to identify cytotoxic effects using in vitro test system
43
2.2 Typical methods to study different aspect of apoptosis 46
2.3 GHS acute oral toxicity classifications 66
3.1 The preparation of different concentrations of gallic acid solution for calibration plot
69
3.2 Preparation of reaction mixture of selected extracts or fractions, DPPH and methanol
71
3.3 Preparation of reaction mixture of selected extracts, DPPH and methanol
72 3.4 Reaction mixture of positive reference standards, DPPH and
methanol for DPPH assay
72
3.5 Reaction components for one-step RT-PCR 87
3.6 Thermal cycling profile of one-step RT-PCR 88
3.7 The sequence of primers used in RT-PCR for preparation of QSTD 89
3.8 The sequence of LUX primers used in qPCR 93
3.9 Reaction components for qPCR 94
3.10 Thermal cycling and melting profile of qPCR 95
3.11 DNA-primer mix preparation for optimization assay 96
3.12 Reaction components for qPCR 97
3.13 Concentrations of Primer-MgCl2 mix for optimization assay 98
3.14 Reaction components for qPCR 98
3.15 Reaction components for cDNA synthesis 101
4.1 Yield of methanol extracts of P. bleo and P. grandifolia 120 4.2 Yield of extracts fractionated from P. bleo crude methanol extract 121
xviii 4.3 Yield of extracts fractionated from P. grandifolia crude methanol
extract
121
4.4 Reducing capacity of P. bleo and P. grandifolia extracts 124 4.5 The scavenging activity (IC50 values) of P. bleo and P. grandifolia
extracts on the inhibition of scavenging activity of DPPH radicals
129 4.6 Reducing powers of extracts of P. bleo and P. grandifolia at various
concentrations
135
4.7 Antioxidant activity (%) of P. bleo and P. grandifolia extracts measured by ß-carotene bleaching method
139 4.8 Results of the antimicrobial tests of the investigated plants in agar
diffusion assay
146
4.9 MIC and MBC of P. bleo and P. grandifolia extracts after 24 h incubation with bacteria
149
4.10 The IC50 values of P. bleo and P. grandifolia extracts tested against KB cell line
156 4.11 The IC50 values of P. bleo and P. grandifolia extracts tested against
CasKi cell line
158
4.12 The IC50 values of P. bleo and P. grandifolia extracts tested against HCT 116 cell line
160 4.13 The IC50 values of P. bleo and P. grandifolia extracts tested against
MCF7 cell line
162
4.14 The IC50 values of P. bleo and P. grandifolia extracts tested against A549 cell line
164
4.15 The IC50 values of P. bleo and P. grandifolia extracts tested against MRC-5 cell line
166 4.16 The IC50 values of doxorubicin against various cancer and non-
cancer cell lines tested
167
4.17 Comparison between IC50 values of P. bleo and P. grandifolia extracts against various cancer and non-cancer cell lines
170 4.18 Comparison of chemistry options for real-time amplification 176 4.19 Results of the potential toxic effect of the crude extracts of P. bleo
and P. grandifolia in mice
199
4.20 Cytotoxic activity (IC50 values) of 2,4-di-tert-butylphenol (1) against 231
xix selected human cell lines
4.21 Cytotoxic activity (IC50 values) of α-tocopherol (2) against selected human cell lines
238
4.22 Cytotoxic activity (IC50 values) of phytol (3) against selected human cell lines
243 4.23 Cytotoxic activity (IC50 values) of ß-sitosterol (4) against selected
human cell lines
244
4.24 Cytotoxic activity (IC50 values) of dihydroactinidiolide (5) against selected human cell lines
246 4.25 Cytotoxic activity (IC50 values) of mixture of sterols (mixture A)
against selected human cell lines
248
4.26 Cytotoxic activity (IC50 values) of phytone (8) against selected human cell lines
250
4.27 Cytotoxic activity (IC50 values) of mixture B against selected human cell lines
255 4.28 Cytotoxic activity (IC50 values) of chemical constituents isolated
from the bioactive ethyl acetate extracts of P. bleo and P.
grandifolia
260
4.29 Cytotoxic activity (IC50 values) of bioactive extracts and chemical constituents isolated from the bioactive ethyl acetate extract of P.
bleo
261
xx LIST OF FIGURES
Figures Page
1.1 Outline of general procedures 7
2.1 The cell cycle 12
2.2 Main steps in apoptosis 14
2.3 The two main routes for triggering apoptosis 16
2.4 Mechanism of apoptosis 18
2.5 The appearance of P. bleo in Seksyen 17, Petaling Jaya, Selangor, Malaysia
25
2.6 The stem of P. bleo 25
2.7 The hemispherical fruit of P. bleo 26
2.8 The orangish-red flowers of P. bleo 26
2.9 The appearance of P. grandifolia in Seksyen 17, Petaling Jaya, Selangor, Malaysia
29
2.10 The stem of P. grandifolia 30
2.11 The pink flowers of P. grandifolia 30
2.12 Alkaloids isolated from P. bleo and P. grandifolia 31 2.13 Compounds isolated from dried powdered fruits of P. grandifolia 32
2.14 Steps in gene expression process 50
2.15 General PCR schema 56
2.16 RT-PCR quantification 57
2.17 The qPCR amplification curve 59
2.18 LUX primer reaction 62
2.19 Melting curve analysis confirming specific amplification with LUX primers
64 3.1 Example of the results of the agar disk diffusion assay 77
xxi 3.2 Induction of apoptosis by the DNase I and DMSO on the KB cells 83
3.3 Example of result analyzed by the software 104
3.4 The extraction and fractionation procedures leading to the isolation of chemical constituents from the bioactive ethyl acetate extract of P. bleo
112
3.5 The extraction and isolation of compounds 1-5 and mixture A from the bioactive ethyl acetate extract of P. bleo
113
3.6 The extraction and fractionation procedures leading to the isolation of chemical constituents from the bioactive ethyl acetate extract of P. grandifolia
116
3.7 The extraction and isolation of compounds 1, 2, 4, 8 and mixture B from the bioactive ethyl acetate extract of P. grandifolia
117
4.1 The gallic acid calibration graph 124
4.2 Reducing capacity of P. bleo and P. grandifolia extracts 125 4.3 The principal of DPPH radical scavenging assay 127 4.4 Scavenging effect of P. bleo extracts on DPPH radical 128 4.5 Scavenging effect of P. grandifolia extracts on DPPH radical 128 4.6 Scavenging effect of positive reference standards (BHA and
ascorbic acid) on DPPH radical
129
4.7 Scavenging effect of lower concentrations of P. bleo extracts on DPPH radical to determine the IC50 values
130 4.8 Scavenging effect of lower concentrations of P. grandifolia extracts
on DPPH radical to determine the IC50 values
131
4.9 Reducing powers of ascorbic acid and BHA (reference compounds) 136 4.10 Reducing powers of extracts of P. bleo at various concentrations 136 4.11 Reducing powers of extracts of P. grandifolia at various
concentrations
137 4.12 Antioxidant activity (%) of P. bleo extracts measured by ß-carotene
bleaching method
140
4.13 Antioxidant activity (%) of P. grandifolia extracts measured by ß- carotene bleaching method
140
xxii 4.14 The in vitro growth inhibitions of KB cells by P. bleo extracts
determined by neutral red cytotoxicity assay
155
4.15 The in vitro growth inhibitions of KB cells by P. grandifolia extracts determined by neutral red cytotoxicity assay
155 4.16 The in vitro growth inhibitions of CasKi cells by P. bleo extracts
determined by neutral red cytotoxicity assay
157
4.17 The in vitro growth inhibitions of CasKi cells by P. grandifolia extracts determined by neutral red cytotoxicity assay
157 4.18 The in vitro growth inhibitions of HCT 116 cells by P. bleo
extracts determined by neutral red cytotoxicity assay
159
4.19 The in vitro growth inhibitions of HCT 116 cells by P. grandifolia extracts determined by neutral red cytotoxicity assay
159
4.20 The in vitro growth inhibitions of MCF7 cells by P. bleo extracts determined by neutral red cytotoxicity assay
161 4.21 The in vitro growth inhibitions of MCF7 cells by P. grandifolia
extracts determined by using neutral red cytotoxicity assay
161
4.22 The in vitro growth inhibitions of A549 cells by P. bleo extracts determined by neutral red cytotoxicity assay
163
4.23 The in vitro growth inhibitions of A549 cells by P. grandifolia extracts determined by neutral red cytotoxicity assay
163 4.24 The in vitro growth inhibitions of MRC-5 cells by P. bleo extracts
determined by neutral red cytotoxicity assay
165
4.25 The in vitro growth inhibitions of MRC-5 cells by P. grandifolia extracts determined by neutral red cytotoxicity assay
165 4.26 The in vitro growth inhibitions of selected human cells by
doxorubicin as positive reference standard determined by neutral red cytotoxicity assay
167
4.27 The specificity of FAM-labelled LUX primers in green channel 179 4.28 The specificity of JOE-labelled LUX primers in yellow channel 180 4.29 Melting curve of FAM-labelled LUX primers in green channel 180 4.30 Melting curve of JOE-labelled LUX primers in yellow channel 181 4.31 The standard curve and amplification curve obtained by the LUX
qPCR assays on the dilution series of ß-actin QSTD template
184
xxiii 4.32 The mRNA expression of p53, caspase-3 and c-myc detected in KB
cells treated with methanol extract of P. bleo for different durations 191
4.33 The mRNA expression of p53, caspase-3 and c-myc detected in KB cells treated with ethyl acetate extract of P. bleo for different durations
192
4.34 The mRNA expression of p53, caspase-3 and c-myc detected in KB cells treated with hexane extract of P. grandifolia for different durations
194
4.35 The mRNA expression of p53, caspase-3 and c-myc detected in KB cells treated with ethyl acetate extract of P. grandifolia for different durations
195
4.36 The mRNA expression of p53, caspase-3 and c-myc detected in MCF 7 cells treated with ethyl acetate extract of P. grandifolia for different durations
196
4.37 Compounds identified from the hexane extract of P. bleo using GCMS analysis
201
4.38 Compounds identified from the hexane extract of P. grandifolia using GCMS analysis
204 4.39 McLafferty rearrangement of methyl palmitate 207 4.40 Chemical constituents from the bioactive ethyl acetate extract of P.
bleo
208
4.41 Chemical constituents from the bioactive ethyl acetate extract of P.
grandifolia
222 4.42 The in vitro growth inhibitions of 2,4-di-tert-butylphenol (1)
against selected human cell lines determined by neutral red cytotoxicity assay
231
4.43 The mRNA expression of p53, caspase-3 and c-myc detected in KB cells treated with 2,4-di-tert-butylphenol (1) for different durations
233
4.44 The mRNA expression of p53, caspase-3 and c-myc detected in MCF7 cells treated with 2,4-di-tert-butylphenol (1) for different durations
234
4.45 The mRNA expression of p53, caspase-3 and c-myc detected in CasKi cells treated with 2,4-di-tert-butylphenol (1) for different durations
235
4.46 The mRNA expression of p53, caspase-3 and c-myc detected in A549 cells treated with 2,4-di-tert-butylphenol (1) for different durations
236
xxiv 4.47 The in vitro growth inhibitions of α-tocopherol (2) against selected
human cell lines determined by neutral red cytotoxicity assay
238
4.48 The mRNA expression of p53, caspase-3 and c-myc detected in CasKi cells treated with α-tocopherol (2) for different durations
240 4.49 The mRNA expression of p53, caspase-3 and c-myc detected in
A549 cells treated with α-tocopherol (2) for different durations
241
4.50 The in vitro growth inhibitions of phytol (3) against selected human cell lines determined by neutral red cytotoxicity assay
242 4.51 The in vitro growth inhibitions of ß-sitosterol (4) against selected
human cell lines determined by neutral red cytotoxicity assay
244
4.52 The in vitro growth inhibitions of dihydroactinidiolide (5) against selected human cell lines determined by neutral red cytotoxicity assay
245
4.53 The mRNA expression of p53, caspase-3 and c-myc detected in HCT 116 cells treated with dihydroactinidiolide (5) for different durations
247
4.54 The in vitro growth inhibitions of mixture of sterols (mixture A) against selected human cell lines determined by neutral red cytotoxicity assay
248
4.55 The in vitro growth inhibitions of phytone (8) against selected human cell lines determined by neutral red cytotoxicity assay
249
4.56 The mRNA expression of p53, caspase-3 and c-myc detected in CasKi cells treated with phytone (8) for different durations
251 4.57 The mRNA expression of caspase-3 and c-myc detected in CasKi
cells treated with phytone (8) for different durations
252
4.58 The mRNA expression of p53, caspase-3 and c-myc detected in HCT 116 cells treated with phytone (8) for different durations
253 4.59 The in vitro growth inhibitions of mixture B against selected
human cell lines determined by neutral red cytotoxicity assay
254
4.60 The mRNA expression of p53, caspase-3 and c-myc detected in CasKi cells cells treated with mixture B for different durations
256
4.61 The mRNA expression of p53, caspase-3 and c-myc detected in A549 cells treated with mixture B for different durations
257
xxv LIST OF APPENDICES
Appendices Page
A Cell culture techniques 297
B Gas chromatogram of hexane extract of P. bleo 301
B1 Mass spectrum of methyl palmitate 301
B2 Mass spectrum of methyl linoleate 302
B3 Mass spectrum of methyl α-linolenate 302
B4 Mass spectrum of phytol 303
C Gas chromatogram of hexane extract of P. grandifolia 304
C1 Mass spectrum of methyl palmitate 304
C2 Mass spectrum of methyl linoleate 305
C3 Mass spectrum of methyl α-linoleate 305
C4 Mass spectrum of methyl stearate 306
D Mass spectrum of 2,4-di-tert-butylphenol (1) 306
D1 1H-NMR spectrum of 2,4-di-tert-butylphenol (1) 307 D2 13C-NMR spectrum of 2,4-di-tert-butylphenol (1) 308
D3 DEPT spectrum of 2,4-di-tert-butylphenol (1) 309
E Mass spectrum of α-tocopherol (2) 310
F Mass spectrum of phytol (3) 310
F1 1H-NMR spectrum of phytol (3) 311
F2 13C-NMR spectrum of phytol (3) 312
F3 DEPT spectrum of phytol (3) 313
G Mass spectrum of ß-sitosterol (4) 314
G1 1H-NMR spectrum of ß-sitosterol (4) 315
G2 13C-NMR spectrum o f ß-sitosterol (4) 316
xxvi
H Mass spectrum of dihydroactinidiolide (5) 317
I Gas chromatogram of mixture A 317
I1 Mass spectrum of campesterol (6) 318
I2 Mass spectrum of stigmasterol (7) 318
I3 Mass spectrum of ß-sitosterol (4) 319
J Mass spectrum of 2,4-di-tert-butylphenol (1) 319
K Mass spectrum of α-tocopherol (2) 320
L Mass spectrum of ß-sitosterol (4) 320
M Mass spectrum of phytone (8) 321
N Gas chromatogram of mixture B 321
N1 Mass spectrum of 2,4-di-tert-butylphenol (1) 322
N2 Mass spectrum of methyl palmitate (9) 322
N3 Mass spectrum of methyl oleate (10) 323
N4 Mass spectrum of methyl stearate (11) 323
O List of publications 324
xxvii LIST OF ABBREVIATIONS
ANOVA ANalysis Of VAriance
ATCC American Tissue Culture Collection A549 Human lung carcinoma cell line BHA
BLAST CasKi CC CDCl3
cDNA
Butylated hydroxyanisole
Basic Local Alignment and Search Tool Human cervical carcinoma cell line Column chromatography
Deuterated chloroform
Complementary deoxyribonucleic acid CH2Cl2 Dichloromethane
CHCl3 Chloroform CO2
Ct
°C DEPC DEPT
Carbon dioxide Threshold cycle Degree Celsius
Diethyl pyrocarbonate
Distortionless Enhancement by Polarisation Transfer DMSO
DNA dNTP
Dimethyl sulfoxide Deoxyribonucleic acid
Deoxyribonucleic triphosphate DPPH
ds
1,1-Diphenyl-2-picrylhydrazyl Double stranded
EDTA Ethylene diamine tetra acetic acid ELISA Enzyme-linked immunosorbent assay EtOAc Ethyl acetate
FAM 6-carboxy-fluorescein FBS Foetal Bovine Serum
g Gram
GCMS Gas Chromatography Mass Spectroscopy HCT 116 Human colon carcinoma cell line
HEPES HPLC h
N-2-Hydroxylethyl-Piperazine-N-2-Ethane-Sulfonoc High Pressured Liquid Chromatography
Hour
xxviii
H2O Water
IC50
IR
Inhibition Concentration at 50 % Infra Red
JOE KB kg
6-carboxy-4’,5’-dichloro-2’,7’-dimethoxy-flurescein Human nasopharyngeal epidermoid carcinoma cell line Kilogram
L LD50
LUX
Litre
Lethal Dose at 50 % Light Upon eXtension
µg Microgram
µl Microlitre
µm Micrometer
MBC Minimum Bacterial Concentration
MCF7 Hormone-dependent breast carcinoma cell line
MeOH Methanol
Mg MgCl2
Milligram
Magnesium chloride
MIC Minimum Inhibitory Concentration
min Minute
ml Millilitre
Mm MRC-5 mRNA MS
Millimetre
Non-cancer human fibroblast cell line Messenger RNA
Mass Spectroscopy NaCl Sodium chloride Na2CO3 Sodium carbonate NaHCO3
NMR
Sodium bicarbonate
Nuclear Magnetic Resonance
nm Nanometre
NRTC Non-reverse-transcriptase control NTC Non-template control
OD Optical Density
PBS PCR
Phosphate buffered saline Polymerase Chain Reaction
xxix Prep-TLC
qPCR QSTD RNA ROS
Preparative-Thin Layer Chromatography Real-time polymerase chain reaction Quantitative standard
Ribonucleic acid
Reactive oxygen species rpm
RT-PCR RT-qPCR s
SD
Rotation per minute
Reverse-transcriptase polymerase chain reaction
Real-time reverse-transcriptase polymerase chain reaction Second
Standard deviation spp.
ss TA
TBE buffer Tm
Taq TLC TUNEL
Species
Single stranded
Annealing temperature Tris-Borate-EDTA buffer Melting temperature Thermus aquaticu
Thin Layer Chromatography
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling
UV Ultraviolet
% Percentage
1 CHAPTER 1
INTRODUCTION
Today, naturally derived products play an important role as source of medicine.
Many pharmaceutical agents have been discovered by screening natural products from plants and marine organisms. The structural diversity of compounds derived from natural products provides valuable sources of novel lead compounds against newly discovered therapeutic targets (Harvey, 1999).
The statistics regarding the uses of natural products as drug are now well-known and have been repeatedly presented and discussed. Over the period 1981-2002, 1031 new chemical entities (NCEs) have been discovered. Thus, in the area of cancer the percentage of new chemical entities that are non-synthetic has remained at 62 % averaged over the whole time frame. In the antihypertensive area, of the 74 formally known synthetic drugs, 48 can be traced to natural product structures/ mimics (Newman et al., 2003; Abas, 2005).
There are around 250,000 plant species in the world and 60 % of them are located in the tropical rainforests. The plant resources of Malaysia comprise about 15,000 species of higher plants. It was estimated that about 1,000 of these plants have undergone simple chemical screening and much less have been subjected to thorough chemical or pharmacological studies (Goh et al., 1993). The huge diversity of the Malaysian flora means that we can expect well diversed chemical structures from their secondary metabolites, and chemical diversity is one of the plus factors that makes natural products excellent candidates for any screening programme.
2 Ethnopharmacological data has been one of the common useful ways for the discovery of biological active compounds from plants, in which the selection of a plant is based on the prior information on the folk medicine use of the plant. It is generally known that ethnomedical data provides substantially increased chance of finding active plants relative to random approach (Chapuis et al., 1988; Cordell et al., 1991).
Pereskia bleo (P. bleo) and Pereskia grandifolia (P. grandifolia), commonly known as the ‘Jarum Tujuh Bilah’ in Malay and ‘Cak Sing Cam’ in Chinese belong to the botanical family Cactaceae. P. bleo can be easily mistaken from P. grandifolia because they look similar vegetatively. However, they can be distinguished by the leaves, flowers and spines. P. bleo have thinner and corrugated leaves, orangish-red flowers with shorter spines compared to P. grandifolia. In contrast, P. grandifolia have thicker and uncorrugated leaves, pink to purple-pink flowers with longer and lesser spines.
Both P. bleo and P. grandifolia have been used as natural remedy in cancer- related diseases, either eaten raw or taken as a concoction brewed from fresh plant. The leaves are also taken as vegetables by some natives. Both are believed to have anti- cancer, anti-tumour, anti-rheumatic, anti-ulcer and anti-inflammatory properties. They are also used as remedy for the relief of headache, gastric pain, ulcers, haemorrhoids and atopic dermatitis; and refresh the body (Goh, 2000; Rahmat, 2004; Tan et al., 2005).
The pounded leaf paste of P. bleo is also applied to the wound or cut for pain relief (Kehidupan Sihat, 2006). In Panama, the locals use the whole plant of P. bleo to treat the gastrointestinal problems (Gupta et al., 1996). On the other hand, P. grandifolia is also used to reduce swellings (Sahu et al., 1974; Anon, 1969).
3 Although P. bleo and P. grandifolia are reported to be used in a large number of Malaysian traditional medicine preparations, there is not much recorded data on biological studies and chemical investigations of P. bleo and P. grandifolia. There is only one phytochemical report (Doetsch et al., 1980) and four biological studies (Matsuse et al., 1999; Tan et al., 2005; Ruegg et al., 2006; Er et al., 2007) reported for P. bleo. Similarly, very little phytochemical work (Doetsch et al., 1980; Sahu et al., 1974) and biological study (Ooi et al., 2003) has been reported for P. grandifolia.
The experimental approach in the present study is based on bioassay-guided fractionation. In this endeavour, the crude methanol and fractionated extracts of P. bleo and P. grandifolia were firstly prepared for the biological assessment. The extracts were subjected to antioxidant assays, antimicrobial assays and neutral red cytotoxicity assay to identify the bioactive extracts of both Pereskia species. The cytotoxic active extracts were further subjected to detection of DNA fragmentation (apoptosis) and determination of the expression level of apoptotic–related genes to verify the possible mechanisms of cell death elicited by the extracts on the cells.
After identifying the bioactive extracts of P. bleo and P. grandifolia, the chemical constituents responsible for the bioactivities were identified. Thus, the bioactive extracts were subjected to isolation and purification procedures to obtain chemical constituents present in the extracts. The isolated chemical constituents were further tested for their cytotoxic activity against the selected human cell lines. The active cytotoxic chemical constituents were then subjected to detection of apoptosis and determination of the expression level of apoptotic–related genes.
Acute oral toxicity was also undertaken in the present study to determine the safety parameters of the leaves of both Pereskia spp. as in vitro trials did not always
4 reflect the outcome of in vivo studies. In addition, the locally grown P. bleo and P.
grandifolia were screened for alkaloids in the present study to confirm the presence of alkaloids as Doetsch et al. (1980) reported the isolation of alkaloids in both Pereskia spp. The general procedures in the present study are outlined in Figure 1.1.
In the present study, the antioxidant activities of the extracts were determined by four different assays, namely scavenging activity of plant extracts on 1, 1-diphenyl- 2-picrylhydrazyl (DPPH) radicals, reducing power assay, ß-carotene method and Folin- Ciocalteau’s method. To our knowledge, there is no antioxidant study reported for both P. bleo and P. grandifolia.
The antimicrobial activities of the extracts were determined by agar diffusion and broth dilution method. The broth dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). To our knowledge, there is only one report on the antimicrobial properties of P.
bleo (Ruegg et al., 2006) and no such report on P. grandifolia.
Determination of cytotoxic effect of Pereskia spp. extracts and isolated chemical constituents were investigated on six human cell lines, which were the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), hormone-dependent breast carcinoma cell line (MCF7), human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5). To our knowledge, no report on the cytotoxicity of P. bleo and P. grandifolia against the above mentioned human cancer cell lines has ever been published.
Apoptosis or programmed cell death plays important roles in many biological processes including carcinogenesis, tumorigenesis and cancer. Many chemotherapeutic
5 and chemopreventive agents have been found to induce apoptotic cell death (Kong et al., 2001). In the present study, DNA fragmentation (apoptosis) was detected using a modified TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling] assay.
Subsequently, the protocol for the evaluation of mRNA expression levels of apoptosis-related genes by LUX RT-qPCR [real-time reverse transcriptase–polymerase chain reaction (RT-qPCR) using LUX (Light Upon eXtension) primers] analysis has been developed in the present study. This LUX RT-qPCR assay offers significant advantages with respect to the rapidity, sensitivity and reproducibility of quantification assay for gene expression.
Objectives of study
The main objectives of the present study were as follows:
a. to screen for antioxidant, antimicrobial and in vitro cytotoxic activities of P.
bleo and P. grandifolia extracts.
b. to investigate the ability of cytotoxic active extracts of P. bleo and P.
grandifolia to induce apoptosis and to determine the possible mechanisms of cell death elicited by the extracts on selected cancer cells.
c. to isolate the chemical constituents from the identified bioactive extracts of P.
bleo and P. grandifolia through the bioassay guided fractionation technique.
d. to identify and elucidate the structures of the chemical constituents by using modern spectroscopic methods.
6 e. to evaluate the cytotoxic activities of the chemical constituents, their ability to induce apoptosis and possible mechanisms of cell death elicited by the chemical constituents on selected cancer cells.
f. to develop a protocol for the evaluation of mRNA expression levels of apoptosis-related genes by LUX RT-qPCR assay [real-time reverse transcriptase–polymerase chain reaction (RT-qPCR) using LUX (Light Upon eXtension) primers].
g. to determine the safety parameters of P. bleo and P. grandifolia extracts by using acute oral toxicity assessment.
7
……continued Fresh leaves of Pereskia spp.
Washed, dried and ground Dried and ground plant material
Extraction with methanol (3x)
Concentration under reduced pressure Methanol extract
Extraction with hexane
Hexane-soluble fraction Hexane-insoluble fraction Partition (v/v) Ethyl acetate: water (1:1)
Ethyl acetate fraction Water fraction
Biological screenings:
1. Antioxidant activity 2. Antimicrobial activity 3. Cytotoxic activity
Detection of DNA fragmentation (apoptosis) If cytotoxic active
If active
Determination of the expression level of apoptotic-related genes
Acute oral toxicity Screening for alkaloids
Identified bioactive fractions GCMS
analysis
8 Figure 1.1, continued
Figure 1.1: Outline of general procedures Identified bioactive fractions
Column chromatography, TLC, prep-TLC and HPLC
Chemical constituents
Spectroscopic and spectrometric identification (NMR, MS) Identified chemical constituents
Screening of cytotoxic activity
If active If active
Determination of the expression level of apoptotic-related genes Detection of DNA fragmentation (apoptosis)
9 CHAPTER 2
LITERATURE REVIEW
2.1 Natural products
Natural products usually refer to secondary metabolites which has relatively complex structures. Secondary metabolites are usually characteristic of specific botanical sources in comparison to primary metabolites which occur in almost every plant. A general characteristic of natural products is that few of them have a clearly recognized function in the metabolic activities of the organisms in which they are found (Geissman et al., 1969).
Natural products can be classified into various families, such as alkaloids, terpenoids and phenolics including flavonoids. Natural products perform various functions, and many of them have interesting and useful biological activities (Harvey, 1999). The utility of natural products as a source of novel structures is still alive and well. The number of plants used as medicinal agents in commerce globally is unknown, but there are at least 1,000 species alone in China (Duke and Ayensu, 1985; Abas, 2005).
2.2 Cancer
Cancer is the general term for a series of neoplastic diseases that are characterized by changes in a cell leading to abnormal (unordered and uncontrolled) cellular proliferation (Pettit, 1997). The disorder occurs in the normal processes of cell division, which are controlled by the genetic material (DNA) of the cell. Cancers may be caused in one of three ways, namely incorrect diet, genetic predisposition, and via the environment (Reddy et al., 2003).
10 To date, mortality that results from the common forms of cancer is still unacceptably high. The Second Report of the National Cancer Registry of Malaysia suggested a total of 23,746 cancer cases were diagnosed among Malaysians in the year 2003, comprising 10,473 males and 13,273 females. The crude rate for males was 97.4 per 100,000 population and 127.6 per 100,000 populations for females. The age standardized incidence rate (ASR) for all cancers was 134.3 per 100,000 males and 154.2 per 100,000 females (The Second Report of National Cancer Registry, 2003).
2.2.1 Carcinogens
The majority of human cancers result from exposure to environmental carcinogens; these include both natural and manmade chemicals, radiation and viruses.
Carcinogens may be divided into several classes, such as (i) genotoxic carcinogens, if they react with nucleic acids. These can be directly acting or primary carcinogens, if they are of such reactivity so as to directly affect cellular constituents; (ii) alternatively, they may be procarcinogens that require metabolic activation to induce carcinogenesis;
(iii) epigenetic carcinogens are those that are not genotoxic (Reddy et al., 2003;
Timbrell, 2000). It is also clear that genetic predisposition is one of the factors of human cancers apart from exposure to carcinogens. Thus, patients with the genetic xeroderma pigmentosum are more susceptible to skin cancer (Reddy et al., 2003).
Carcinogens in the diet that trigger the initial stage include moulds and aflatoxins (for example, in peanuts and maize), nitrosamines (in smoked meats and other cured products), rancid fats and cooking oils, alcohol, and additives and preservatives. A combination of foods may have a cumulative effect, and when incorrect diet is added to a polluted environment, smoking, UV radiation, free radicals,
11 lack of exercise, and stress, the stage is set for DNA damage and cancer progression (Reddy et al., 2003).
2.2.2 Cell cycle
In cells that are dividing, the nuclear DNA molecules must be duplicated and then distributed in a way that ensures the two new cells receive a complete set of genetic instructions. The cells pass through a series of discrete stages called G1 phase, S phase, G2 phase and M phase in order to accomplish these tasks (Kleinsmith, 2006).
These four phases are collectively referred to as the cell cycle.
The cell cycle is commonly represented by a circular diagram (Figure 2.1). The G1, S and G2 phases are collectively referred as interphase. Besides providing the time needed for a cell to make copies of its DNA molecules, interphase is also a period of cell growth. Interphase occupies about 95 % of a typical cell cycle; whereas the actual process of cell division (M phase) only takes about 5 % (Kleinsmith, 2006). G1 is defined as the interval between M phase and S phase, and G2 is defined as the interval between S phase and M phase. S phase is defined as the time during the cell cycle when DNA synthesis is taking place, leading to a doubling of the amount of DNA per cell. M phase is the time when the amount of DNA per cell drops in half as cells divide. The restriction point is a control point near the end of G1 where the cell cycle can be halted until conditions are suitable for progression into S phase. Under normal conditions, the ability to pass through the restriction point is governed mainly by the presence of growth factors.
12 Figure 2.1: The cell cycle
(Adapted from http://herb4cancer.files.wordpress.com/2007/11/cell-cycle2.jpg, 19 August 2009)
2.2.3 Carcinogenesis
The transformation of a normal cell into a cancerous cell is believed to proceed through many stages over a number of years or even decades. Carcinogenesis is a multistep process that involves initiation, promotion and tumour progression. Initiation involves a reaction between the cancer-producing substance (carcinogen) and the DNA of tissue cells. There may be a genetic susceptibility. This stage may remain dormant, and the subject may only be at risk for developing cancer at a later stage. Promotion involves a prolonged period of proliferation of the initiated cells, occurs very slowly over a period ranging from several months to years. During this stage, a change in diet and lifestyle can have a beneficial effect so that the person may not develop cancer during his or her lifetime. The third and final stage involves progression and spread of the cancer, at which point diet may have less of an impact. Preventing initiation is an important anticancer strategy, as are the opportunities to inhibit cancer throughout the latter stages of malignancy (Reddy et al., 2003; Kleinsmith, 2006).
Restriction point
13 2.3 Apoptosis in cancer
Most of the cells from higher eukaryotes have the ability to self-destruct by activation of an intrinsic cellular suicide program referred to as programmed cell death or apoptosis (Ellis et al., 1991; Steller, 1995). Apoptosis is an intrinsic biological event that plays an essential role in development, homeostasis and in many disease process.
Culling extra cells in a precise and systematic way is an important aspect of normal development. Hence, the other term for this process is programmed cell death.
Occasionally, cell death program goes awry in several diseases. Degenerative diseases may result in (or be the result of) excessive apoptosis and some cancers appear to inhibit cell death cascades resulting in excessive and uncontrolled proliferation (O’Brien et al., 1998).
Apoptosis is a unique type of cell death, different from what happens when cells are destroyed by physical injury or exposure to certain poisons. In response to such non-specific damage, cells undergo necrosis, a slow type of death in which cells swell and eventually burst, spewing their contents into surrounding tissues. Necrosis often results in a local inflammatory reaction that can cause further cell destruction, which makes it potentially dangerous (Kleinsmith, 2006; O’Brien et al., 1998).
In contrast, apoptosis kills cells quickly and neatly, without causing damage to surrounding tissue. Apoptosis is characterized by certain morphological features, including reduction in cell volume, cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation (Kerr et al., 1972, 1994; Wyllie et al., 1980;
O’Brien et al., 1998). The process involves a carefully orchestrated sequence of intracellular events that systematically dismantle the cell (Figure 2.2).
14 Figure 2.2: Main steps in apoptosis (Kleinsmith, 2006)
As a cell begins to undergo apoptosis, the first observable change is cell shrinkage. Next, small bubble-like protrusions of cytoplasm form at the cell membrane as the nucleus and other cellular structures begin to disintegrate. The chromosomal DNA is then degraded into small pieces and the entire cell breaks apart, forming small fragments known as membrane-bound apoptotic bodies that are engulfed by macrophages or neighbouring phagocytic cells without generating an inflammatory response. Phagocytic cells are specialized for ingesting foreign matter and breaking it down into molecules that can be recycled for other purposes (Kleinsmith, 2006).
The genetic basis for apoptosis implies that cell death, like any other metabolic or development program, can be disrupted by mutation. Elucidation of the core machinery of apoptosis has provided new insights into cancer biology, revealing novel strategies for cancer therapy (Reed, 2002). In the last decade, basic cancer research has produced remarkable advances in the understanding of cancer biology and cancer genetics. Among the most important of these advances is the realization that apoptosis and the genes that control it have a profound effect on the malignant phenotype. For example, it is now clear that some oncogenic mutations disrupt apoptosis, leading to tumour initiation, progression or metastasis. Conversely, compelling evidence indicates